The invention is related to the field of multiphoton excitation microscopy/microfabrication, and in particular to axial resolution for two-photon wide-field illumination microscopy and microfabrication.
Multiphoton excitation fluorescence microscopy has recently gained popularity for cellular and tissue imaging. It provides intrinsic three-dimensional (3-D) resolution, allows deep imaging into tissues, achieves submicron optical resolution, and minimizes photodamage and photobleaching. Moreover, multiphoton excitation microfabrication has been also widely used since it can generate finer 3-D features than conventional two-dimensional (2-D) lithographic techniques. However, both systems use either laser scanning or laser writing technique to achieve this intrinsic optical sectioning capability based on spatially focusing laser light at the focal point of a high numerical aperture objective. It is common to implement laser scanning microscopy and laser writing microfabrication, but its major drawback is the longer image acquisition or fabrication time than that in the wide-field illumination based systems. This limits multiphoton fabrication in producing small prototypes although the submicron optical resolution and optical sectioning capabilities are very attractive for making 3-D structures.
Recently, the concept of temporal focusing was introduced, which disperses optical pulse into monochromatic waves at different angles on a grating surface and recombine them at focal plane. It is very useful in multiphoton depth-resolved wide-field illumination, since the original optical pulses are restored only at focal plane, and several applications in the nonlinear microscopy were proposed. However, temporal focusing has never been applied to 3-D lithographic microfabrication. In addition, depth discrimination capability for wide-field illumination system has not been fully evaluated both theoretically and empirically although it is one of the most important parameter in designing multiphoton wide-field illumination systems.